Extracellular levels of glutamate are controlled with great precision both temporally and spatially, achieving efficient and selective synaptic excitation and preventing excitotoxic neuronal death. On one hand, glutamate concentrations in the synaptic cleft must rise rapidly to millimolar concentrations to ensure activation of postsynaptic ionotropic receptors. On the other hand, the average extracellular concentration of glutamate must be maintained at sub-micromolar levels to prevent cell death. These requirements are met by the explosive exocytotic release of glutamate and the high capacity and high affinity glutamate uptake system provided by the family of Na-dependent glutamate transporters. When transporter function is compromised experimentally or by metabolic crises such as ischemia, elevated tonic levels of glutamate and slowing of glutamate clearance around synaptic release sites can result in seizures, enhanced spreading damage from ischemic insults and neuronal and organismal death. The objective of this proposal is to determine how much glutamate escapes from the synaptic cleft following release, how far from the release site glutamate reaches concentrations sufficient to activate receptors, how rapidly the uptake system sequesters glutamate, and how these processes are affected by physiological alterations in the amount of glutamate released including multivesicular release. We will investigate these issues at three dissimilar synapses to compare how their unique morphologies and expression patterns of receptors and transporters affect the actions of glutamate released synaptically and applied exogenously by the photolysis of caged glutamate. For these studies, we will use patch clamp recordings in conjunction with two photon laser scanning microscopy and two photon laser glutamate uncaging in acute slices of rat cerebellum and hippocampus. By pairing electrical and optical recording we achieve both high temporal and spatial resolution. Two photon laser glutamate uncaging is particular well suited to studying diffusion of glutamate released from individual synapses because short applications (0.5 ms) of high concentrations (mM) in small volumes (~ 1 m3) can be achieved. Such applications approach those of vesicular exocytosis but are much more easily controlled experimentally. Using this technique as well as synaptic release, we will address the consequences of ionotropic and metabotropic glutamate receptor activation inside and outside of the synaptic cleft. PUBLIC HEALTH RELEVANCE: Understanding how the brain functions depends on knowledge of the fundamental unit of information processing within the brain called the synapse. To understand the synapse, we need detailed information about its physical structure, molecular constituents, biochemical and physiological mechanisms and how these properties change with age and experience. Rational design of therapies for neurological deficits is not possible without a fundamental understanding of synaptic function.